The present invention relates to new thermostable DNA polymerases derived from archaeobacteria of the genus Pyrococcus sp.
The DNA polymerases are enzymes involved in the replication and repair of DNA. There are currently numerous known DNA polymerases isolated from microorganisms such as E. [Escherichia] coli; for example, DNA polymerase I of E. coli, the Klenow fragment of DNA polymerase I of E. coli, DNA polymerase T4. Thermostable DNA polymerases are also identified and purified from thermophilic organisms, such as Thermus aquaticus (Chien, A., et al., J. Bacteriol. 1976, 127:1550-1557; Kaladin et al., Biokhymiyay 1980, 45:644-651), Thermus thermophilus, or else the species bacillus (European Patent Application No. 699,760), thermococcus (European Patent Application No. 455,430), sulfobus and pyrococcus (European Patent Application No. 547,359). Among these, it is possible to mention more particularly Pfu of Pyrococcus furiosus, the Vent DNA polymerase of Thermococcus litoralis (Kong, H. M., R. B. Kucera, and W. E. Jack, 1993, J. Biol. Chem. 268(3):1965-1975), 9xc2x0-NDNA polymerase of Pyrococcus sp., 9xc2x0-N, and Deep-Vent DNA polymerase of Pyrococcus sp. GB-D.
The replication process takes place according to a well-known mechanism comprising manufacturing (from a template, DNA polymerase enzyme and four triphosphate nucleotides) a strand of complementary nucleic acid of said template. The enzymes with DNA polymerase activity are currently widely used in vitro in numerous molecular biology processes, such as cloning, detection, labeling, and amplification of nucleic acid sequences.
Amplification of nucleic acid sequences by the method called polymerase chain reaction (PCR), described in the European Patent Nos. 200,362 and 201,184, is based on the execution of successive cycles of extensions of primers, using a DNA polymerase and the four triphosphate nucleotides, followed by denaturation of the double-strand nucleic acids thus obtained and used as templates for the next cycle. Since the temperatures used in the denaturation step are not compatible with preservation of the activities of numerous DNA polymerases, important research studies are dedicated to the thermostable enzymes described in the preceding. It is particularly essential not to limit the preparation of these enzymes solely to the processes of purification from microorganisms, but to seek to increase the production yields using methods of genetic engineering. According to these methods, which are well known to the experts in the field (Maniatis et al., Molecular Cloning: A Laboratory Manual, 1982), the gene coding for DNA polymerase is cloned in an expression vector, a vector which is inserted in a cellular host capable of expressing the enzyme, the cellular host is grown under suitable conditions, and the DNA polymerase is isolated and recovered. This method was described, for example, in the patent application PCT WO89/06691 for producing DNA polymerase of Thermus aquaticus. 
The present invention relates to thermostable purified DNA polymerase of archaeobacteria of the genus Pyrococcus sp.